Masaaki Soma

Eicosapentaenoic acid inhibits vasopressin-activated Ca2+ influx and cell proliferation in rat aortic smooth muscle cell lines

The purpose of this study was to clarify how eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid, modulates the vascular action of vasopressin in rat aortic smooth muscle cell lines. The effects of EPA on Ca2+ mobilization and DNA synthesis elicited by vasopressin were investigated and compared to those of Ca2+ channel blocking agents, by means of Ca2+ measurements and the incorporation of [3H]thymidine. Patch-clamp techniques were also employed. Vasopressin (100 nM) elicited an initial peak of intracellular Ca2+ ([Ca2+]i), followed by a sustained phase due to Ca2+ entry. Nifedipine or nicardipine (1 microM), a potent L-type Ca2+ channel blocker, partly inhibited the sustained phase, but La3+ completely abolished it. EPA (10 microM) also inhibited it even in the presence of nicardipine. Under voltage-clamp conditions with CsCl-internal solution, depolarizing pulses positive to -30 mV from a holding potential of -40 mV elicited a slow inward current. The inward current was blocked by La3+, nicardipine, and nifedipine (1 microM), suggesting that the inward current mainly consisted of the voltage-dependent L-type Ca2+ channel (ICa.L). EPA (1-30 microM) also inhibited ICa.L in a concentration-dependent manner. The inhibitory effect of EPA was observed at concentrations higher than 1 microM, and its half-maximal inhibitory concentration (IC50) was 7.6 microM. Vasopressin induced a long-lasting inward current at a holding potential of -40 mV. The vasopressin-induced current was considered as a non-selective cation current (Icat) with a reversal potential of approximately +0 mV. Both nifedipine and nicardipine (10 microM) failed to inhibit it significantly, but La3+ completely abolished Icat. EPA also inhibited vasopressin-induced Icat in a concentration-dependent manner; its IC50 value was 5.9 microM. Vasopressin (100 nM) stimulated [3H]thymidine incorporation. Exclusion of extracellular Ca2+ with EGTA or La3+ markedly inhibited it. EPA (3-30 microM) also inhibited the incorporation induced by vasopressin, while nifedipine and nicardipine (1 microM) only partly inhibited it. These results suggested that EPA, unlike nifedipine and nicardipine, inhibited vasopressin-induced Ca2+-entry and proliferation in rat vascular smooth muscle cells, where the inhibitory effects of EPA on Icat as well as ICa.L might be involved. Thus, EPA would exert hypotensive and antiatherosclerotic effects.

EPA effect on NOS gene expression and on NO level in endothelin-1-induced hypertrophied cardiomyocytes.

Cardiomyocytes release (or metabolize) several diffusible agents (e.g., nitric oxide [NO], endothelin-1 [ET-1], and angiotensin II) that exert direct effects on myocyte function under various pathologic conditions. Although cardiac hypertrophy is a compensatory mechanism in response to different cardiovascular diseases, there can be a pathologic transition in which the myocardium becomes dysfunctional. Recently, NO has been found to be an important regulator of cardiac remodeling. Specifically, NO has been recognized as a potent antihypertrophic and proapoptotic mediator in cultured cardiomyocytes. We demonstrated that ET-1–induced hypertrophic remodeling in neonatal cardiomyocytes was arrested by pretreatment with eicosapentaenoic acid (EPA), a major component of fish oil. In some recent studies, EPA has demonstrated cardioprotective effects by modulating NO. This study investigated the changes in NO synthase (NOS) in ET-1–induced hypertrophied cardiomyocytes and in total levels of nitrates and nitrites. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured in D-MEM/Ham F12 supplemented with 0.1% fatty acid–free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control group, ET-1 (0.1 nM) group, and ET-1 pretreated with EPA (10 μM) group. NOS gene expression was evaluated 24 hrs after treatment using real-time polymerase chain reaction. Endothelial NOS (eNOS) mRNA expression was decreased in the ET-1 group compared with controls and was unchanged by pretreatment with EPA. mRNA expression of inducible NOS (iNOS) was significantly increased in ET-1–treated cardiomyocytes and was suppressed by EPA pretreatment. Neuronal NOS gene expression and total NO level did not exhibit a statistically significant change in any of the groups. There may be some interaction between ET-1, eNOS, and iNOS in ET-1–induced and EPA-regressed hypertrophied cardiomyocytes that suppress iNOS expression without modulating total NO level or eNOS gene expression.

ω-3 Polyunsaturated fatty acids—modulation of voltage-dependent L-type Ca2+ current in guinea-pig tracheal smooth muscle cells

Abstract ω-3 polyunsaturated fatty acids have been reported to be associated with favorable changes in the respiratory system. To determine one of the mechanisms for this effect, membrane currents were recorded in guinea-pig tracheal myocytes by using the whole-cell voltage clamp technique. Without EGTA in the patch pipette containing the Cs-internal solution, command voltage pulses positive to +0 mV from a holding potential of −60 mV elicited a voltage-dependent L-type Ca 2+ current ( I Ca·L ) and a subsequent outward current. Upon repolarization, slowly decaying inward tail currents were recorded. The outward currents and the inward tail current were enhanced by methyl-1,4,-dihydro-2,6-dimethyl-3-nitro-4-(2-trigluromethylphenyl)-pyridine-5-carboxylate, and blocked by Cd 2+ or nifedipine. Inclusion of EGTA (5 mM) in the patch pipette also abolished these currents, indicating that they were Ca 2+ -dependent. When [Cl − ] o or [Cl − ] i was changed, the reversal potential of these currents shifted, thus behaving like a Cl − -sensitive ion channel. 4,4′-Diisothiocyanatostilbene-2,2′-disulphonic acid, a Cl − channel blocker, inhibited the currents. The ω-3 polyunsaturated fatty acids eicosapentaenoic acid (3–30 μM) and docosahexaenoic acid (30 μM) suppressed I Ca·L and then inhibited I Ca·Cl in a reversible manner. Similar inhibitory effects of eicosapentaenoic acid on I Ca·L were observed with 5 mM EGTA in the patch pipette. Neurokinin A (1 μM) and caffeine (10 mM) also transiently activated I Cl·Ca , probably due to Ca 2+ release from Ca 2+ storage sites. Pretreatment of the cells with eicosapentaenoic acid markedly suppressed the activation of I Cl·Ca by neurokinin A or caffeine. These results suggest that ω-3 polyunsaturated fatty acids inhibit voltage-dependent L-type Ca 2+ currents and also Ca 2+ -activated Cl − currents in tracheal smooth muscle cells from the guinea-pig, which may play a role in modulation of tracheal smooth muscle tone.

Changes in important apoptosis-related molecules in the endothelin-1-induced hypertrophied cardiomyocytes: effect of the pretreatment with eicosapentaenoic Acid.

Human heart failure is preceded by a process called cardiac remodeling, in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Endothelin (ET)-1, a potent vasoconstrictor peptide, is synthesized and secreted by cardiomyocytes and induces hypertrophy of cardiomyocytes. The cardiovascular benefit of fish oil containing eicosapentaenoic acid (EPA) in humans and experimental animals was reported. Recently, we found that ET-1–induced cardiomyocytic remodeling could be prevented by pretreatment with EPA. The aim of the present study is to investigate whether there would be any alteration in the expression of important apoptosis-related molecules in ET-1–administered hypertrophied cardiomyocytes. We also sought to determine, if there are alterations in apoptotic molecules, what type of role for EPA would then exist. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control, the ET-1 (0.1 nM)–treated group, and the ET-1 group pretreated with EPA (10 μM). Twenty-four hours after the treatment, the gene expressions of three important molecules related to apoptosis (caspase-3, Bax, and Bcl-2) in three experimental groups were evaluated by real-time polymerase chain reaction. The present study could not demonstrate any significant or representative alteration in any of the above three apoptosis-related important markers in either ET-1–induced hypertrophied cardiomyocytes with or without EPA pretreatment. The present study would at least be able to exclude the involvement of some representative molecules related to apoptosis in ET-1–induced hypertrophied cardiomyocytes. In addition, the present study demonstrates that the antihypertrophic effect of EPA to ET-1–administered cardiomyocytes appears not to modulate the apoptosis signaling cascade.

Eicosapentaenoic acid enhances myo-inositol uptake in cultured human aortic smooth muscle cells

The effects of elevated glucose and Eicosapentaenoic acid (EPA, 20:5) on myoinositol uptake in human aortic smooth muscle cells (HASMC) were evaluated. Myo-inositol incorporation into HASMC was dependent on an active transport system via Na(+)-K+ ATPase activity based on the results with Na+ deprivation and Ouabain (5 mM). Although glucose (27.5, 55 mM) inhibited 2-[3H] myo-inositol uptake, the addition of EPA (3 x 10(-4) M) prevented glucose-mediated inhibition. In addition, EPA potentiated Na(+)-K+ ATPase activity of HASMC. Since EPA decrease glucose-mediated inhibition of myo-inositol uptake, this agent might ameliorate aortic smooth muscle cell function associated with diabetes.

Effect of Eicosapentaenoic Acid on the Different Endothelin System Components in Endothelin-1–Induced Hypertrophied Cardiomyocytes

The cardiovascular benefit of fish oil, including eicosapentaenoic acid (EPA), in humans and experimental animals has been reported. The role of endothelin-1 (ET-1) in cardiac hypertrophy is well known. Endothelin-1 stimulates prepro–ET-1 mRNA expression in cardiomyocytes, and the autocrine/paracrine system of ET-1 is important for cardiomyocyte hypertrophy. Although many studies link EPA to cardiac protection, the effect of EPA on cardiac hypertrophy has yet to be clarified. Recently, we demonstrated that ET-1–induced cardiomyocytic change could be prevented by pretreatment with EPA. The present study investigated the changes of different components of the ET system at the mRNA level in ET-1–administered cardiomyocytes, and examined the effect of EPA pretreatment. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats, cultured in Dulbecco’s modified Eagle’s medium and Ham F12 supplemented with 0.1% fatty acid–free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into 3 groups: control group, ET-1–treated (0.1 nM) group, and ET-1–treated group pretreated with EPA (10 μM). Twenty-four hours after treatment, the gene expressions of different components of the endothelin system in three experimental groups were evaluated by real-time polymerase chain reaction. Prepro–ET-1 mRNA expression was 53% upregulated in ET-1–induced hypertrophied cardiomyocytes and suppressed in the EPA-pretreated group. Endothelin-converting enzyme-1 (ECE-1) was also increased in ET-1–administered cardiomyocytes by 42% compared with the control group and was reversed in the EPA-pretreated group. The two receptors of ET system, ETA and ETB, tended to be increased in the ET-1–treated group, but no statistical significance was seen among study groups. Endothelin-1 increased prepro–ET-1 and ECE-1 mRNA expression in hypertrophied-neonatal cardiomyocytes, and this was reversed with EPA pretreatment. Thus, EPA may play a crucial role in the regression of ET-1–induced cardiomyocyte hypertrophy, partly through the suppression of ET-1 and ECE-1 expression.